I collected data from the Sony SH800S flow cytometer, however, I am seeing an inconsistency with the data from two different runs. During both runs, I gated for CD11b and CD45 co-positive cells and was trying to evaluate the percent of parent population of my target on this population. Using wildtype control mice of same sex and age, the first run showed 50% and the next run was about 98%. In addition, on the second run, all of the treatment groups had this extremely high percent. We eliminated the possibility of protocol, antibody, and concentration changes, the panels matched, and I was the one who ran both runs. The only change I did make was a new compensation matrix. Could this one change make that significant of a difference? If not, has anyone seen this before or have suggestions on what this could be? The inconsistency in the data is a concern and we are trying to figure out the source, so any help is very much appreciated!
Were the runs on the same day ? And if so y did you change the compensation matrix ? If not, did you use any calibration beads or cst beads ? Did the qc pass ?
Compensation matric changes can produce some degree of inconsistency but not to the grade you have described provided all other conditions are kept same.
Raghumoy Ghosh The runs were not on the same day and so we did change the compensation matrix. Yes, we used calibration beads and the qc passed.
We are also confused on why this would have resulted in such high numbers since there was minimal change between the runs. I also gated using FMOs, so I would assume the FMOs would have reflected any changes from the matrix.
Dear Amanda Leisgang
The effect of compensation matrix can be predicted if you could tell the fluorochroms of your antibodies. Compensation matrix make big changes in readings.
If your QC is passed, FMO are run then it may be rectifiable.
Will you mind showing your plots?
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